The present invention relates to methods, computer readable storage media and systems for analyzing labeled biological samples such as fluorescence in situ hybridization (FISH)—stained samples, such as for identifying chromosomal aberrations, for identifying genetically abnormal cells and for computational scanning of samples.
Various pathologies such as infections, inflammation, cancer and/or genetic diseases are currently diagnosed by analyzing microscopic slides containing biological specimens such as blood samples, urine sample, tissue biopsies, spinal fluid aspirates, amniotic fluid, chorionic villi sample (CVS) and the like. Since most of the biological samples are transparent, the first step of analysis requires staining with a specific dye or probe which enables the detection of almost any tissue or cellular specimen constituent. Such constituents may be, for example, a connective tissue, a cell organelle (e.g. mitochondria), a certain protein or DNA sequence, a specific chromosomal region and/or a specific RNA transcript. Common staining methods include cytological staining (e.g., Giemsa, Hematoxylin), immunological staining (e.g., immunohistochemistry, immunofluorescence), activity staining (e.g., using fluorescent or chromogenic substrates), cytogenetic staining (e.g., G-banding or R-banding), in situ chromosomal staining (e.g., using FISH) and/or in situ DNA staining [e.g., using quantitative-FISH (Q-FISH)]. The dyes or probes used in such staining methods can be detected using bright-field microscopy with transmitted light, fluorescence microscopy or both. Most of the staining methods utilize commercial kits with all necessary chemicals and stains for labeling and detection. Once the sample is stained, it can be viewed using a microscope or an imaging system either manually by eye observation or automatically using a computer-controlled analysis system. Most imaging systems are based on charged coupled device (CCD) camera detectors that are controlled by a computer and appropriate acquisition control and image analysis software. Other imaging systems use a point detector such as photomultiplier (PMT) and a focused laser source and scan the image point-by-point.
The first step in applying these imaging systems involves the identification of regions of interests (ROIs) on the microscopic slide. In many research and clinical applications, the user manually scans the microscopic slide and then captures the ROIs for further analysis. However, since manual identification of ROIs is a time- and labor-consuming effort, which complicates the overall analysis process, systems for automatically scanning and analyzing biological samples have been developed. Such systems usually combine a microscope setup, an imaging device such as a CCD camera together with a computer-controlled hardware that allows scanning of the sample in three dimensions (3D) with high accuracy and precision [see for example, H. Netten et al., 1997, Cytometry 28: 1-10 or the CytoVision SPOT system by Applied Imaging Corporation, San Jose Calif., USA)]. A typical automated system includes optical elements (e.g., a color filter), a control over stage movement, which enables the scanning of all desired areas, and a correlation of stage motion with image detection (using e.g., the CCD detector). Such an automated system also requires the ascertainment of the correct focus (i.e., the z-position) of the sample so that the detected object is in the correct focal plane of the microscope and the selection of the optimal exposure time of the camera and of other acquisition parameters.
Because of the large area that has to be scanned on the microscope slide and the typically high resolution that is needed, many images have to be scanned from each slide. This requires a certain strategy for scanning the relevant slide-area in an efficient way. Currently practiced methods of scanning the ROIs begin with the center of slide and proceed in a circular manner outward, moving from one ROI to an adjacent one. However, if the cells to be scanned are not evenly distributed on the slide, scanning may begin with an area that is completely irrelevant for analysis, thus resulting in a considerable waist of scanning time until reaching the areas containing cells. In addition, in many cases, the cells of interest (e.g., cancerous cells), which are part of a tissue sample, are not positioned in the center of the specimen, thus leading to either mis-diagnosis due to under-scanned areas or to enormously long scanning times which are impractical for both research and clinical applications. To date, there is no available method which can efficiently select ROIs in a reliable and time-consuming manner.
The detection of multiple fluorescent probes and automation of analysis is of significant advantage in the application of fluorescence in situ hybridization (FISH). FISH is employed to map the precise location of genes on chromosomes, and to detect very small genetic defects not visible by gross staining of chromosomes (e.g., by G-banding or R-banding). Thus, FISH staining is widely used for detection of genetic aberrations at the chromosome level (e.g., chromosome amplification, deletion, translocation, rearrangement) [H. Netten et al., FISH and Chips: Automation of Fluorescent Dot Counting in Interphase Cell Nuclei. Cytometry 28: 1-10 (1997)]. Many FISH applications merely require the cytogeneticist to look through the eyepieces of a microscope, or at the image on the monitor, and to determine whether a fluorescent label is present or absent. With somewhat more complex specimens, a simple count of one or two colored labels may be done (e.g., for determination of gender using probes from the X and Y chromosomes). Other applications require the enumeration of different fluorescent signals in each cell and the determination of a ratio therebetween. For example, the amplification of the Her-2 gene, which is used in staging and/or determining the prognosis of stage II, node-positive, breast cancer patients, can be detected using the PathVision kit (Vysis Inc. Chicago Ill., USA). Such a kit includes a FISH probe for the Her-2 gene (labeled with a SpectrumOrange™ fluorochrome) and a FISH probe for the centromere of chromosome 17 (SpectrumGreen™). Other FISH probes can be used to detect various translocations by detecting merged fluorescent signals. For example, the BCR/abl ES kit (e.g. Vysis Inc. Chicago Ill., USA) can be used to diagnose the presence of a Philadelphia chromosome, which is associated with chronic myeloid leukemia (CML), by detecting the presence of merged fluorescent signals resulting from fusion of chromosomes 9 and 22. Although chromosomal translocations may also result in reduced intensity of staining of one stained spot as compared to another spot labeled with the same probe, as was unintentionally shown in the case of the unbalanced translocation involving chromosomes 18 and 20 [t(18; 20)(p11.1; p11.1)] (Czako M., et al., 2002 (Am. J. Med. Genet. 108: 226-8)), currently practiced FISH analysis methods do not include and/or rely on the relative intensity measurements of the same probe as part of a routine FISH analysis.
For most clinical and research applications, the FISH results are being interpreted automatically using image analysis systems. One of the main problems associated with the currently available automated image analysis techniques is the presence of split, merged or overlapping spots resulting from either technical reasons or associated with the presence of translocations and/or sub-deletions which are difficult to interpret. For example, a merged spot of two different probes (labeled with different fluorescent dyes) can result from either a real translocation of the two separate chromosome sequences, or from an accidental close proximity of these two chromosome sequences (that is known to happen occasionally). In addition, a single spot on a single chromosome locus can appear as a split-spot with two close-by spots if the observed chromosome contains already two copies of the genome (after the S-phase of the cell cycle) and the two chromatids of the chromosome are somewhat far apart. In addition, stain debris may show up as extra spots which can mistakenly be counted as additional “real” spots. Moreover, the hybridization conditions may be sub-optimal, thus resulting in non-specific binding of probe and extra background stain which may lead to false analysis of the image.
Existing imaging systems currently include either a monochrome camera or a color camera for image analysis. While bright field and simple FISH probes with only 1-3 colors can be imaged and analyzed in a single exposure using a color camera, most of FISH and especially multi-color FISH probes need to be imaged with multiple acquisitions through different excitation and emission color filters using a monochrome camera. A color camera has three color filters that would usually not match the emission spectral ranges of the fluorochromes, and therefore a monochrome camera is much more favorable. Thus, if the imaging system includes only a color camera, it can result in poor quality and signal to noise measurements of the weak fluorescent staining. On the other hand, if the imaging system includes a monochrome camera, measurements of the bright signals, which are often performed using a bright field mode, but sometimes also using a fluorescent mode, are time-consuming and complicated.
There is thus a widely recognized need for, and it would be highly advantageous to have, image analysis methods and systems devoid of the above limitations.